Project Summary Zika virus (ZIKV) is a recently recognized global health threat due to the wide geographic distribution of risk and potential for severe consequences of infection. There remains no antiviral in clinical use for ZIKV, or any flavivirus, and many unanswered questions regarding the basic biology of these viruses. Our preliminary data suggest that IRE1?, a component of the cellular response to endoplasmic reticulum (ER) stress, is required for ZIKV replication. IRE1? also promotes optimal replication of flaviviruses related to ZIKV via an unknown mechanism. Based on these data, we hypothesize that IRE1? inhibitors may be therapeutic against flaviviral infection. This proposal aims to assess IRE1? activation in ZIKV infected cells, understand the molecular mechanism(s) by which IRE1? supports ZIKV replication and determine whether targeting IRE1? could be efficacious in a mouse model of infection. IRE1? is activated during the unfolded protein response, a cellular pathway to detect and alleviate dysfunctional protein folding in the ER. During ER stress, IRE1? initiates nonconventional splicing of XBP1 mRNA. Spliced XBP1 encodes a transcription factor, which upregulates targets involved in ER function. IRE1? has more recently been shown to target other specific RNAs leading to their degradation. IRE1? inhibitors are under evaluation for treatment of non-infectious human diseases, and we propose that this project will provide preclinical evidence for the novel application of these drugs to treat flaviviral infection. The experiments outlined in this proposal will extend our preliminary findings to important primary cell types and currently circulating ZIKV strains (Aim 1). We will also determine whether the requirement for IRE1? in ZIKV infection is XBP1-dependent or -independent, which will direct experiments to dissect the downstream cellular processes required for ZIKV infection. In addition, we will identify the stage(s) of ZIKV replication supported by IRE1? by systematically examining viral binding, RNA replication, protein synthesis and ER remodeling (Aim 2). We predict that IRE1? most likely supports biogenesis of ER-derived viral replication platforms, and will focus experiments on this hypothesis. Finally, we will determine whether IRE1? could be targeted as an antiviral therapeutic strategy in mouse models of ZIKV infection (Aim 3). Together these aims will reveal basic and practical insights into the biology of ZIKV replication, which are likely relevant for other flaviviruses including established and potentially emergent human pathogens.